中国兽医杂志2025，Vol.61Issue(11)：64-69,6.DOI:10.20157/j.cnki.zgsyzz.2025.sf.005
猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立
Establishment of Double-Antibody Sandwich ELISA Detection Method for Porcine Epidemic Diarrhea Virus
摘要
Abstract
Porcine epidemic diarrhea virus(PEDV)is one of the major pathogens causing diarrhea in piglets,posing a serious threat to the pig industry.In this study,a double-antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA)was developed for PEDV detection using a monoclonal antibody(mAb)against the PEDV N protein as the capture antibody and a rabbit-derived polyclonal antibody(pAb)as the detection antibody.Optimal assay conditions were determined,and the specificity,sensitivity,and reproducibility of the method were evaluated.Additionally,the consistency between this method and reverse transcription polymerase chain reaction(RT-PCR)was assessed using 96 clinical samples.The optimal reaction conditions were as follows:coating with 0.478 mg/L mAb at 4℃for 12 h,blocking with 3%BSA at 37℃for 60 min,incubation with virus samples at 37℃for 20 min,incubation with 3.75 mg/L rabbit anti-PEDV pAb at 37℃for 45 min,and incubation with horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG(diluted 1∶5 000)at 37℃for 30 min.The developed ELISA showed no cross-reactivity with PRRSV,TGEV,PDCoV,or PCV,indicating high specificity.The detection limit was 1.65×10² TCID₅₀/mL(4.7×10⁻⁴ mg/mL),demonstrating high sensitivity.The intra-and inter-assay coefficients of variation ranged from 3.3%to 5.9%and from 3.0%to 6.2%,respectively,indicating good reproducibility.The positive concordance rate with RT-PCR was 91.67%(22/24),the negative concordance rate was 98.61%(71/72),and the overall concordance rate was 96.88%(93/96).These results indicate that the developed DAS-ELISA is highly specific,sensitive,and reproducible,and is suitable for clinical detection of PEDV.关键词
猪流行性腹泻病毒(PEDV)/单克隆抗体(mAb)/多克隆抗体(pAb)/双抗体夹心酶联免疫吸附试验(DAS-ELISA)Key words
porcine epidemic diarrhea virus(PEDV)/monoclonal antibody(mAb)/polyclonal antibody(pAb)/DAS-ELISA分类
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曹文艳,江佳欣,袁子钦,王亚楠,王自良,王磊,李春艳,李任峰..猪流行性腹泻病毒双抗体夹心ELISA检测方法的建立[J].中国兽医杂志,2025,61(11):64-69,6.基金项目
河南省重点研发与推广专项(科技攻关)农业领域基金资助项目(242102111010) （科技攻关）
